DESIGN RULES FOR COACERVATE-BASED EMULSIONS ENABLING ENZYME CATALYSIS IN ORGANIC SOLVENTS

Complex coacervates have been extensively investigated for protein encapsulation, providing a dense, polymer-rich microenvironment that enhances enzyme stability and modulates catalytic performance in aqueous systems. However, their potential for biocatalysis remains largely unexplored in non-aqueous environments, particularly in reactions involving hydrophobic substrates. In such cases, organic solvents are often required, but these conditions typically lead to enzyme destabilization and reduced activity. Here, we present a strategy to stabilize enzyme-loaded coacervate droplets in water-immiscible organic solvents through the formation of highly stable emulsions. The emulsions are prepared from poly(diallyldimethylammonium hydroxide) (PDADMAOH) and poly(acrylic acid) (PAA) coacervates, stabilized by a polystyrene-based amphiphilic anionic random copolymer, and dispersed in solvents including toluene, chlorobenzene, chloroform, and dichloromethane. The resulting microdroplets exhibit exceptional resistance to coalescence, maintain integrity under centrifugation, and remain stable for weeks, enabling straightforward separation and redispersion for repeated use. Using α-chymotrypsin as a model enzyme and a fluorogenic peptide substrate (N-succinyl-Ala-Ala-Pro-Phe-7-amido-4-methylcoumarin), we demonstrate that the aqueous interior of the droplets preserves enzyme structure and catalytic activity in non-aqueous media. Beyond establishing the feasibility of this approach, we systematically investigate how the amount of water retained within the coacervate phase influences enzymatic activity. Our findings define a set of design rules for developing coacervate-based platforms for biocatalysis in organic media, including guidelines on polymer composition, selection of amphiphilic stabilizers, solvent compatibility, and control of water content. By enabling recyclable, enzyme-loaded aqueous microdomains to operate within non-aqueous environments, this work broadens the applicability of coacervate systems to industrially relevant transformations involving poorly water-soluble substrates. This framework offers a versatile and tunable route for extending coacervate-mediated catalysis far beyond the constraints of traditional aqueous-phase reactions, paving the way for customizable and sustainable enzyme immobilization strategies in challenging chemical environments.